Journal:

Article Title: Genetically altered AMPA-type glutamate receptor kinetics in interneurons disrupt long-range synchrony of gamma oscillation

doi: 10.1073/pnas.051631898

Figure Lengend Snippet: Generation of mutant mice with elevated GluR-B expression in GABAergic interneurons. (a) Schematic representation of gene segments of the wild-type GAD67 allele, the targeting vector, and the targeted GAD67 allele. Positions of EcoRI (E), NotI (N), and SalI (S) restriction sites are indicated. The PCR fragment used as probe for the Southern blot is indicated as a black bar. (Right) Southern blot analysis of tail DNA isolated from wild-type and heterozygous mice, digested with EcoRI to distinguish wild-type (7-kb) and targeted (5-kb) alleles. (b–e) GluR-B immunostaining in wild-type (b and d) and combined immunostaining and X-Gal staining in the mutant (c and e) hippocampus. Note the much higher GluR-B expression in LacZ-positive GABAergic interneurons compared with neighboring pyramidal cells in the mutant CA1 region (c) and hilus (e). This expression was never seen in the corresponding areas in control animals (b and d). (Scale bars, 100 μm.) so, Stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; h, hilus. For higher magnification see Fig. 6 in the supplemental data.

Article Snippet: After X-Gal staining, the free-floating sections were incubated overnight at 4°C with a rabbit anti-GluR-B antibody (PharMingen, dilution 1:25) in 2% normal goat serum diluted in Tris-buffered saline (TBS), pH 7.4.

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Southern Blot, Isolation, Immunostaining, Staining

Journal:

Article Title: Genetically altered AMPA-type glutamate receptor kinetics in interneurons disrupt long-range synchrony of gamma oscillation

doi: 10.1073/pnas.051631898

Figure Lengend Snippet: Firing properties of interneurons are altered by genetically manipulated overexpression of GluR-B. (a) Spontaneous EPSP rise time and decay time plots for stratum pyramidale fast-spiking interneurons, recorded from a membrane potential of −70 mV. Data are shown as cumulative probability for >4,000 EPSPs from five cells from five wild-type and five cells from five mutant mice. Model data show change in unitary EPSC for parameters that best fit the experimental data: “wild-type” unitary EPSC = t exp (−t/0.95) nS, “mutant” unitary EPSC = t exp(−t/1.05) nS. (Scale bars: 0.8 nS, 1 ms.) (b) Firing pattern in response to a single proximal stratum radiatum stimulation. Traces show response (from −70 mV) to increasing stimulus intensity (5–20 V) for interneurons from wild-type and mutant. Note increasing intensities generate double spikes in the cell from the mutant. (Scale bars: 50 mV, 40 ms.) Model data show voltage responses for single interneurons (holding current −0.165 nA) receiving identical EPSC onto single compartments on each of three dendrites. Each EPSC had the time course ct exp(−t/τ), where τ = 0.95 for control and 1.05 for mutant; c = 16.1, 47.4, 83.8 nS (top to bottom). Note the spike doublet in the mutant, but not in the wild type (as in experiment). (Scale bars: 50 mV, 30 ms.) (c) Pattern of pyramidal cell phasic inhibitory input (upper traces) and interneuron phasic excitatory input (lower traces) during posttetanic oscillations, in wild-type and in mutant mice. The interneuron was hyperpolarized by injection of −0.2 nA current. [Scale bars (experiment): 2 mV, 100 ms.] Model data show GABAA conductance to an e-cell (upper traces) and AMPA conductance to an i-cell. Note, in the “mutant,” the more variable amplitude of GABAA inputs, and the variable width of the AMPA inputs. [Scale bars (model): 150 nS, 100 ms.] (d) Example traces of interneuron firing patterns during gamma-frequency oscillations following paired tetanic stimulation, illustrating the increased incidence of doublet formation and the occurrence of occasional spike bursts. Model data show voltage of a selected interneuron from network simulations in the wild type and the mutant. The only difference in parameters for these simulations was in interneuron EPSC time course illustrated in a. [Scale bars (experiment and model): 20 mV, 100 ms.] Below are histograms illustrating the probability of a doublet of interval x relative to the sample mode. There are 45 doublets from wild-type data (n = three cells from three animals) and 130 doublets from mutant data (n = five cells from five animals). Model histograms were constructed by using ≈110 doublet intervals for simulations of “wild type” and “mutant” gamma oscillations, each pooled from seven interneurons.

Article Snippet: After X-Gal staining, the free-floating sections were incubated overnight at 4°C with a rabbit anti-GluR-B antibody (PharMingen, dilution 1:25) in 2% normal goat serum diluted in Tris-buffered saline (TBS), pH 7.4.

Techniques: Over Expression, Mutagenesis, Injection, Construct

Journal: Molecular Vision

Article Title: Effect of curcumin on the modulation of αA- and αB-crystallin and heat shock protein 70 in selenium-induced cataractogenesis in Wistar rat pups

doi:

Figure Lengend Snippet: Effects of curcumin on immunohistochemistry of αA- and αB-crystallin in the eye lens of Wistar rat pups exposed to selenium. A : This image shows the control lens incubated in saline alone without any antibody treatment. B : This is a control lens that was incubated with saline and subjected to antibody treatment. C : Lens from rat pups administered with selenium alone. D : Lens from rat pups administered with selenium and curcumin simultaneously. E : Lens from rat pups administered with selenium first and then treated with curcumin after 24 h. F : Lens from rat pups pretreated with curcumin and then administered with selenium after 24 h. Lens sections were preincubated with αA- and αB-crystallin polyclonal antirabbit Immunoglobulin G (IgG) antibody (1:3,000 dilution) and subsequently with goat anti-rabbit IgG-horse radish peroxidase (HRP) conjugate (1:3,000 dilution). The immunoreactivity was developed with 0.01% 3,3-diaminobenzidine tetrahydrochloride (DAB) and H 2 O 2 . Note the brown color formation indicative of peroxidase reaction in the nucleus. Image G , negative control, lens treated with goat anti-rabbit IgG-HRP and developed using DAB and hydrogen peroxide (H 2 O 2 ). The figure shows the high level of αA- and αB-crystallin expression induced by selenium-mediated oxidative stress. This increased crystallin expression and aggregate formation was prevented by curcumin pretreatment.

Article Snippet: After washing with TBS containing 0.05% Tween 20, the sections were incubated with the primary antibody, αA- and αB-crystallin polyclonal anti-rabbit IgG antibody (BD Biosciences, San Jose, CA), at a dilution of 1:500 overnight at 4 °C.

Techniques: Immunohistochemistry, Incubation, Negative Control, Expressing

Journal: Molecular Vision

Article Title: Effect of curcumin on the modulation of αA- and αB-crystallin and heat shock protein 70 in selenium-induced cataractogenesis in Wistar rat pups

doi:

Figure Lengend Snippet: Immunoblot expression of αA- and αB-crystallin in control and experimental group of animals. Lane I, eye lens protein from control (physiologic saline) rat pups (group I); lane II, eye lens protein from selenium-injected rat pups (group II); lane III, eye lens protein from rat pups administered selenium and curcumin simultaneously (group III); lane IV, eye lens protein from rat pups injected with selenium 24 h before being administered with curcumin (group IV); and lane V, eye lens protein from rat pups administered with curcumin 24 h before being injected with selenium (group V). The separated lens protein was preincubated with αA- and αB-crystallin polyclonal antirabbit IgG antibody (1:3,000 dilution) and subsequently with goat antirabbit IgG-HRP (1:3,000 dilution). The immunoreactivity was developed with 0.01% DAB and H 2 O 2 . β-Actin refers to house keeping protein expression and its levels are constant across all treatment groups indicating the normal behaviour of lenses under various treatment. The figure clearly shows increased αA- and αB-crystallin protein expression under selenium-mediated oxidative stress. This increased crystallin protein expression was prevented by curcumin pretreatment.

Article Snippet: After washing with TBS containing 0.05% Tween 20, the sections were incubated with the primary antibody, αA- and αB-crystallin polyclonal anti-rabbit IgG antibody (BD Biosciences, San Jose, CA), at a dilution of 1:500 overnight at 4 °C.

Techniques: Western Blot, Expressing, Injection